Spinal anesthesia using small amounts of tetrodotoxin or desoxytetrodotoxin

ABSTRACT

Vertebrates can be anesthetized regionally by a subarachnoid administration of tetrodotoxin or desoxytetrodotoxin. The amount of tetrodotoxin which should be administered in a preferred embodiment ranges between 1 and 12 micrograms, whereas the amount of desoxytetrodotoxin ranges between 10 and 120 micrograms.

United States Patent Adams et al.

Aug. 5, 1975 SPINAL ANESTHESIA USING SMALL AMOUNTS OF TETRODOTOXIN ORDESOXYTETRODOTOXIN Inventors: Herbert J. F. Adams, Westboro;

Murray R. Blair, Jr., Sudhury; Robert N. Boyes, Auburn; Maxim I.Lebeaux, Shrewsbury; Helen G. Vassall, Worcester, all of Mass AstraPharmaceutical Products, Inc., Worcester, Muss.

Filed: Aug. 14. I973 Appl. No.: 388.147

Assignee:

US. Cl 424/251; 424/!05 Int. Cl A61k 27/00 Field of Search .r 424/105,251

References Cited OTHER PUBLICATIONS Chemical Abstracts, Vol. 77 (1972),p. l68634y.

Primary Exuml'ner-V. D. Turner Attorney Agent, or Firm-Brumbaugh,Graves, Donohue & Raymond [57] ABSTRACT 5 Claims, 1 Drawing FigureSPINAL ANESTHESIA USING SMALL AMOUNTS OF TETRODOTOXIN ORDESOXYTETRODOTOXIN The present invention relates to a method ofobtaining spinal anesthesia (i.e., by injection of an anesthetic intothe subarachnoid space which contains the cerebro-spinal fluid) and apharmaceutical preparation which can be used with this method.

Toxins from marine sources of extraordinary potency have been known formany years. This invention particularly concerns novel uses fortetrodotoxin, and for its derivative, desoxytetrodotoxin.

Tetrodotoxin is obtained from the ovaries and eggs of several species ofpuffer fish of the suborder Gymnoa'onles. It is also found in certainspecies of California newts of the genus Taricha; and the toxin obtainedfrom these species, known as tarichatoxin, is identical withtetrodotoxin. Tetrodotoxin has been purified, and its molecularstructure is determined to be an amino perhydroquinazoline of theformula:

Tetrodotoxin and species in which it occurs are more fully described inP/mrnuuulugical Reviews, Vol. 18, No. 2, at pages 997-1049. Some of theeffects of tetrodotoxin and its derivatives, e.g., desoxytetrodotoxin,are described in Chemical Abstracts. Vol. 37, p. l77O (I943) and Vol.69, Column 9494w (1968).

Experiments with isolated nerves have shown that tetrodotoxin behaves ina fundamentally different manner from local anesthetics such as procaineand cocaine. In a voltage-clamped giant axon from the squid or lobster,the latter agents reduce both inward initial sodium current and outwardpotassium current. With tetrodotoxin, however, inward sodium current canbe reduced or even obliterated, while the outward potassium current istotally unaffected. There are few if any other substances in which thisunique action has been established.

Tetrodotoxin has not heretofore found any practical use as ananesthetic. While the compound can be used to induce nerve blocks inlaboratory animals, the anes thetic dose is slightly below the lethaldose, which has precluded, as a practical matter, the use of the compound as an anesthetic in its own right. M. H. Evans, Tuxicon, I968,Vol. 5, pp. 289294. tested its use topically on peripheral nervebranches and exposed dorsal and ventral spinal roots in anesthetizedcats, and found blocks in the latter case but concluded that theimpermcability of the connective tissue sheath of peripheral nervesseemed likely to preclude its use as a local anesthetic agent.

Combinations of tetrodotoxin with a local anesthetic compound have beenfound to possess unusual anesthetic properties. This is manifested mostsignificantly in improved longevity of action of combinations of thetoxin with local anesthetics. ln these combinations. tetrodotoxin istypically used in concentrations below those which produce reliablenerve blocks. The site of administration of the tetrodotoxin andanesthetic is outside the dura of the spinal canal, i.e., a periduraladministration. It is theorized that the local anesthetic en ables thetetrodotoxin or desoxytetrodotoxin to penetrate the sheath surroundingthe nerve roots and produce local anesthesia.

The present invention relies upon a subarachnoid spinal administrationinto the intact mammal of a composition which consists essentially ofsmall amounts of tetrodotoxin or desoxytetrodotoxin. In this case, it isnot therefore necessary to combine these compounds with (Zwitter'ionform) a local anesthetic, and it has been found that very low doses ofthe compounds give a longer duration of anesthesia than is obtainablewith the use of certain prior art anesthetics of differing composition.For example. it is possible to administer a very low dose of 1-1 2micrograms (pg) of tetrodotoxin or 10-120 micrograms ofdesoxytetrodotoxin and obtain a spinal block of between 3 and 24 hoursin duration. Thus, subarachnoid anesthesia in mammals, including man, iscontem plated. in the case of man, it is believed, based on animaltests, that a minimum dose of l #g of tetrodotoxin is effective toproduce anesthesiav By contrast, subarachnoid administration oftetracaine (at larger doses of 3-20 mg) gives a block of from 1.5 to 3hours and xylocainc (at mush larger doses of SO-ISO mg) gives blocks offrom l to 2 hoursv P. C. Lund. Principles and Practice of SpinalAnesthesia, pp. 420 and 425, Charles C. Thomas (publisher), l97l. Theseknown spinal anesthetics cause more irritation and have a greater localtoxicity than the composition used in the present invention.

It has been found that the administration of the compounds used in thepresent invention should be deliv ered into the subarachnoid area of thespinal column for effective anesthesia to be induced. A particularlypreferred dosage range is from 3 to 6 micrograms of tetrodotoxin and 30to 60 micrograms of desoxytetrodotoxin.

procedure to be performed.

The following examples show the invention in practice:

EXAMPLE I water as the vehicle for the drug. lsobaric solutions are 5usually prepared by using cerebrospinal fluid as the vehicle but sodiumchloride added in a suitable concen- This example illustratesPharmaceutical P tration to an aqueous solution of the compound cantiOnS and methods for P p g thcm which 3T6 yp also be used to obtainisobaricity. Hyperbaric solutions baric Solutions Containing 1 can beprepared by addition of varying amounts of glulo and Mg/ tClfOdOlOXiflhy r h ide; p cose (usually 5lO%) to a water solution of the com pounds.A hyperbaric solution, the most commonly The tetrodotoxin hydrochlorideis dissolved in a Concentration [Lg/ml l 2 l 4 5 s 8 10 Component AmountTetrodotoxin hydrochloride (mg) 10 I00 Hydrochloric acid.

Sodium hydroxide.

Waiter for injection USP XVIII If necessary to adjust pH If necessary toadjust pH Sufficient quantity to make l0 liters small amount of waterand is diluted to a volume of 9.5 liters. The pH is adjusted and asufficient volume of water is added to make a solution of l0 literswhich is then sterilized.

EXAMPLE 2 This example illustrates pharmaceutical composiand thereby theextent of anesthesia in a manner that 30 lions and methods for preparingthem which are isois well known in the art. The acidity of the solutionsdebaric solutions containing 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, scribed aboveis kept inside limits that give good stabil- 8.0, and 100 ug/mltetrodotoxin hydrochloride; pH ity to the tetrodotoxin ordesoxytetrodotoxin. 3,5 5

Concentration Lg/ml l 2 3 4 5 6 8 10 Component Amount Tetrodotoxin I0 203O 40 50 60 80 100 hydrochloride (mg) Sodium chloride 90 90 9o 90 90 9090 USP XVII (gj Hydrochloric acid,

If necessary to adjust pH Sodium hydroxide,

If necessary to adjust pH Water for injection USP XVIII Sufi'icientquantity to make l0 liters.

If desired. addition of a vasoconstrictor, e.g. epinephrine, to thepharmaceutical preparation can also be made in manners that are wellknown in the art. The concentration of active ingredient is generally1-1 2 mi crograms/ml for tetrodotoxin and 10-120 miero- 50 andthereafter a sufficient volume of water is added to grams/ml ofdesoxytetrodotoxin and volumes of apmake a solution of 10 liters whichis then sterilized. proximately l-6 ml of solution are injected into thece- EXAMPLE 3 rebrospmal fluid contained in the subarachnoid space Thisexample illustrates pharmaceutical composi of the vertebrate to beanesthetized. The type of solugs ions 211nd methods for pmparing than.which are tion to be used, the concentration and volume to be adperbuflcsojumms Containing L0 20, 30 40 50 8'0 ministered are dependent on suchfactors as, for lll and Lg/m] tetmdmoxin hydrochloride. pH stance ageand size of the patient and on the surgical Concentration ug/ml l 2 1 4i 5 8 10 Component Amount Tetrodotoxin I (l 20 30 40 50 50 80 I00hydrochloride (mgl Dextrose,

anhydrous 500 500 500 500 500 750 500 500 USP XVIII (gl Hydrochloricacid. 2N If necessary to adjust pH Sodium hydroxide,

Water for injection USP XVIII The tetrodotoxin hydrochloride isdissolved in a small amount of water and diluted to 9.0 liters. The pHis adjusted. The dextrose is added and dissolved. A suf ficient volumeof water is added to make a solution of liters which is then sterilized.

Other forms of tetrodotoxin such as salts of other acids thanhydrochloric acid or such as the base or zwitterion form can be used andsolutions can be pre pared containing amounts equivalent to those oftetro dotoxin hydrochloride given in Examples l3.

In the case of desoxytetrodotoxin. compositions are made in the samemanner as shown in Example l3 for tetrodotoxin. except that theconcentrations of biotoxin are l0 to I00 #g/ml, becausedesoxytctrodotoxin is about one-tenth as active as tetrodotoxin.

minutes of the injection with recovery occurring before I7, I95 and 23hours had elapsed from the time of the injection, respectively. in theanimals. In two animals full recovery occurred within 28 hours, while inthe third some ataxia was noted for an additional [2 to 24 hours withfull recovery within 48 hours from the time of injection. The spread ofthe anesthesia towards the head (ccphalad spread) was on the average of4 to 5 segments from the point of injection. Excessive cephalad spreadis indicated by signs of Horners syndrome. head drop. abdominalbreathing, cyanosis and front limb paresis. None of these signs wereobserved. Anesthesia in the lower region of the spinal column (caudalregion) was complete. Table I sets forth the results obtained.

Table l Spinal Anesthesia in Sheep Drug: Tetrodotoxin Dose: 3 pg 0l.: 1ml. Injection site: L6-Sl Duration Vulva or Spontane Anal ScrotumDigital our Muscle Full Sheep Wt. Onset Re Re Re- Activity Weight Re-No. Sex (k) (min) flex Pain flex Pain flex Pain Rear Leg Support covery190 190 l90 l90 190 190 190 I F 29.3 4 90 min. hr.

hr. hr. hr. hr. hr. hr. hr.

420 420 420 420 420 420 420 2 M 28.9 4 60 min. 28 hr.

hr. hr. hr. hr. hr. hr. hr.

320 320 320 320 320 320 26 hr. 3 M 29.3 4 200 min. 195

Notes Time "I minutes except where indicated. Carrier: 7.5'1 dextrose insaline H=t.0|

EXAMPLE 4 EXAMPLE 5 Three sheep weighing between 28 and 29.5 kg and ofapproximately 4.5 to 5 months of age were treated with tetrodotoxin (3micrograms). All three sheep were restrained with their lumbar andthoracic spine in horizontal position during the subarachnoid injection.which was made over a period of 5 to l0 seconds using a 22 gauge-3 inchspinal needle at the interspace between the sixth lumbar and firstsacral segments. The sheep were then immediately tilted so that theslope of the lumbar and thoracic spine was positioned 10 to thehorizontal (caudal inferior).

The response to the tetrodotoxin was a rapid onset of anesthesia (withinless than 4 minutes, usually within 2 to 3 minutes), with a long sensoryblock followed by complete recovery. Although the limitations of thenormal working day interfered with exact measurements of the duration ofthe block, it was determined that the sensory blocks were still presentafter 190. 320 and 420 The procedure used in Example 4 was employedusing a 6 microgram dose of tetrodotoxin. In the three sheep treatedwith this dose level. the onset of the block was rapid. The blockslasted for longer than 6.5 hours but less than 24 hours. In one case theblock lasted exactly 24 hours. Weight support required 48-72 hours forrecovery and complete recovery usually required 72-86 hours. One animalhad a persistent slight impairment of one joint that lasted 18 days butwhich eventually returned to normal. This was due to inflammation fromcauses other than drug-related causes. Cephalad segmental spread wasslightly more exaggerated than the spread observed when animals weretreated with 3 micrograms. Regression of two segments required 2-3 hourson the average. The Table given below summarizes the test results:

Table II Tetrodotoxin: 6 pg/ml (total dose 6 pg) VoL: l.(] ml. DurationSpontaneous heap Onset Anal Vulval-Scrotal Digital Rear-leg Weight FullWt. Time Re- Pain Reflex Pain Reflex Pain Motor Activity SupportRecovery flex No. 4 6 fi /z 6& 24 hr. 24 hr. 24 hr. 48 hr. 72 hr.

hr. hr. hr. hr. Fe- 3 min.

male

24 hr. 24 hr. 24 hr. 24 hr. 48 hr. 48 hr. 48 hr. 8.2k No. 5 l0 hr. l0hr. l0 hr. l0 hr. l0 hr. I0 hr. l(J hr. 72 hr. 72 hr. Fe- 3-5 male min24 hr. 24 hr. 24 hr. 24 hr. 24 hr. 24 hr. 24 hr. 96 hr.

Table ll-Continued Tetrodotoxini 6 pg/ml (total dose 6 .Lg] Vol. |.U ml.

Duration Spontaneous heep Onset Anal Vulval'ScrotaI Digital Rear'legll/eight Full Wt. Time Pain Reflex Pain Reflex Pain Motor ActivitySupport Recovery No. Slight Fe- 3 min. 24 hr. 24 hr. 24 hr. 24 hr. 26hr. 26 hr. 24 hr. 72 hr. knuckling male right fet- 6.8k

lock- I 8 days Not drug related Nu untoward systemic side cficcts werenoted EXAMPLE 6 l5 scissa or X axis of FIG. I, the anesthesia wears offbeginning antcriorly (cephalad) (because less drug has A l2 microgramdose was tested on one sheep weighreached these nerves) and progressespostcriorly (cauing 5L8 kg. Table III sets forth the results: dally)until total recovery occurs. Each point value in Table III Tetrodotoxin:I2 ug/ml (total dose I2 #gj Vol.1 1.0 ml. Duration Sheep AnalVuIvaI-Scrotal Digital Spontaneous Onset 4 Rear-leg Weight Full WeightTime Reflex Pain Reflex Pam Reflex Pain Motor Activity Support RecoveryNo. 7 10 hr. 10 hr. 10 hr. 10 hr. l0 hr. 48 hr. 96 hr.

3 min. 24 hr. 24 hr. 5 [.Bkg. hr. 20 hr. 20 hr. 20 hr.

FIG. 1 is a graph showing the spread and effect of the w FIG. Itherefore represents the level of anesthesia at a subarachnoidallyadministered local anesthetic, versus I particular time and also thetotal number of spinal segtime after administration. ments which areblocked at that time. FIG. 1 compares Using the technique described inthe foregoing Exthe level of anesthesia and the segmental regressionamples 4-6, the local anesthetic drug to be tested was pattern obtainedin sheep after administration of each injected into the subarachnoidspace (which contains 39 of the three local anesthetics. Tetracainc, themost the cerebrospinal fluid), between the six lumbar and commonly usedspinal anesthetic in humans, in these the first sacral vertebralinterspace of the sheep. Each experiments anesthetized only a total of 6or 7 segof three animals received I.() ml of 0.25% tetracaine or mentsand regressed (the animal recovered) com- 0.5% tetracaine or 1.0 mlcontaining 6 (4g of tetrodopletely in between 3-5 hours. Tctrodotoxin,on the toxin. The most commonly used concentration of tetra- 40 otherhand, at concentrations far below those of tetracaine in spinalanesthesia, which is the most commonly caine blocked a total of between9-12 segments for at used anesthetic in humans, as indicated by Lund(prinleast 6 hours and did not regress completely for about cipals andPractice of Spinal Anesthesia, C. C. Thomas 24 hours.

Publishers, Springfield, Ill., (I971) p. 420) is 0.25% It is observedfrom these experiments, as illustrated 0.3%. Above this concentrationthe drug becomes ingraphically in FIG. I, that tctracaine atconcentrations crcasingly irritating and is not generally used. In manof 0.25 and 0.5 percent would provide anesthesia and the injection isusually made between either the second pain relief in segments below 5-3for only about 3 and third or third and fourth lumbar interspaccs. Thehours. Tetrodotoxin. however, provided anesthesia and drug, afterinjection, mixes with the spinal fluid and is pain relief below 5-3 forat least I5 hours.

carried or spread anteriorly and postcriorly. Recovery of function inthe animals tested was com- On the Y" axis (the ordinate) of FIG. I is ascheplete and no scquelac followed the long duration of anmaticrepresentation of the segments of the spinal cord. B izl CuUSCd ylrodotoxin.

In the sheep, caudal to cephalad (posterior to anterior) Upon readingthe foregoing description, persons in the segments of the spinal cordarc: coccygcal 3,2,]; the art will become aware of a numberofmodiflcations sacral 4,31,]; lumber 6,5,4,3 2,l; thora i that can bemade to the invention described herein I3,l2,l l,l0,9,8,7,6, 5,4,3,2,I;cervical 7,65,43,11. without the exercise of inventive skill. Thesemodifica- Thoracic 13 is the only one of the last two groups illustionsare intended to be included within the scope of trated or represented inFIG. 1. the invention. and the foregoing is intended to be Depending onmany factors such as on ent i merely illustrative otccrtain preferredembodiments of volume of solution, manipulation of the patient and in nothe im'em'mn Th6 pp Claims dQlmL the Scope of herent characteristics ofthe drug, a variable degree of Pmmctlon Soughtduration and spread ofanesthesia can be achieved. We claim:

This spread and its recovery or so-callcd regression is I. A process forinducing spinal anesthesia in a mampictorially represented as in FIG. 1.Above (or cepha mail comprising administration by injection into the ladto) each point there is no anesthesia and the spinal SU bllfaClmO'lClPQC f the intact mammal from about nervgs um ff md b th d B l d l to] lto 6 milliliters of a pharmaceutical composition comeach point allspinal segments or nerves are blocked. prising a compatible vehicle andas the active ingredi- Over a period of time, which is represented onthe abcnt a compound selected from the group consisting of tetroclotoxinin concentration of l to l2 micrograms per milliliter anddesoxytetrodotoxin in concentration from 10 to I micrograms permilliliter of the vehicle.

2. The process as claimed in claim 1 wherein the amount of tetrodotoxinwhich is administered is from about 1 to l2 micrograms.

3. The process as claimed in claim I wherein the amount ofdesoxytctrodotoxin which is administered is

1. A PROCESS FOR INDUCING SPINAL ANESTHESIA IN A MAMMAL COMPRISINGADMINISTRATION BY INJECTION INTO THE SUBARACHNOID SPACE OF THE INTACTMAMMAL FROM ABOUT 1 TO 6 MILLITERS OF A PHARMACEUTICAL COMPOSITIONCOMPRISING A COMPATIBLE VEHICLE AND AS THE ACTIVE INGREDENT ACOMPOUNSELECTED FROM THE GROUP CONSISTING OF TETRODOTOXIN IN CONCENTRATION OF 1TO 12 MICROGRAMS PER MILLITER AND DESOXYTETRODOTOXIN IN CONCERNTRATIONFROM 10 TO 120 MICROGRAMS PER MILLITER OF THE VEHICLE.
 2. The process asclaimed in claim 1 wherein the amount of tetrodotoxin which isadministered is from about 1 to 12 micrograms.
 3. The process as claimedin claim 1 wherein the amount of desoxytetrodotoxin which isadministered is from 10 to 120 micrograms.
 4. The process as claimed inclaim 1 wherein the amount of tetrodotoxin which is administered is fromabout 3 to 6 micrograms.
 5. The process as claimed in claim 1 whereinthe amount of desoxytetrodotoxin which is administered is from about 30to 60 micrograms.